Selective inhibition of Cl(-) conductance in toad skin by blockers of Cl(-) channels and transporters

We compared the effects exerted by two classes of Cl(-) transport inhibitors on a Cl(-)-selective, passive anion transport route across the skin of Bufo viridis, the conductance (G(Cl)) of which can be activated by transepithelial voltage perturbation or high cAMP at short circuit. Inhibitors of antiporters (erythrosine, eosin) or cotransporters (furosemide) reduced voltage-activated G(Cl) with IC(50) of 6 +/- 1, 54 +/- 12, and 607 +/- 125 microM, respectively; they had no effect on the cAMP-induced G(Cl). The voltage for half-maximal activation of G(Cl) (V(50)) increased compared with controls, but effects on the maximal G(Cl) at more positive clamp potentials were small. Cl(-) channel blockers from the diphenylamino-2-carboxylic acid (DPC) family [dichloro-DPC, niflumic acid, flufenamic acid, and 5-nitro-2-(3-phenylpropylamino)benzoic acid] reduced the voltage-activated G(Cl) with IC(50) of 8.3 +/- 1.2, 10.5 +/- 0.6, 16.5 +/- 3.4, and 36.5 +/- 11.4 microM, respectively, and also inhibited the cAMP-induced G(Cl), albeit with slightly larger IC(50). V(50) was not significantly changed compared with controls; the maximal G(Cl) was strongly reduced. We conclude that the pathway for Cl(-) is composed of the conductive pore proper, which is blocked by the derivatives of DPC, and a separate, voltage-sensitive regulator, which is influenced by blockers of cotransporters or antiporters. This influence is partly overcome by increasing the clamp potential and removed by high concentrations of cAMP, which renders the pathway insensitive to voltage.     

pH and external Ca(2+) regulation of a small conductance Cl(-) channel in kidney distal tubule

A single channel characterization of the Cl(-) channels in distal nephron was undertaken using vesicles prepared from plasma membranes of isolated rabbit distal tubules. The presence in this vesicle preparation of ClC-K type Cl(-) channels was first established by immunodetection using an antibody raised against ClC-K isoforms. A ClC-K1 based functional characterization was next performed by investigating the pH and external Ca(2+) regulation of a small conductance Cl(-) channel which we identified previously by channel incorporation experiments. Acidification of the cis (external) solution from pH 7.4 to 6.5 led to a dose-dependent inhibition of the channel open probability P(O). Similarly, changing the trans pH from 7.4 to 6.8 resulted in a 4-fold decrease of the channel P(O) with no effect on the channel conductance. Channel activity also appeared to be regulated by cis (external) Ca(2+) concentration, with a dose-dependent increase in channel activity as a function of the cis Ca(2+) concentration. It is concluded on the basis of these results that the small conductance Cl(-) channel present in rabbit distal tubules is functionally equivalent to the ClC-K1 channel in the rat. In addition, the present work constitutes the first single channel evidence for a chloride channel regulated by external Ca(2+).     

Scalariform junctions in the malpighian tubules of the insects Rhodnius prolixus (Hemiptera: Reduviidae) and Aedes taeniorhynchus (Diptera: Culicidae)

The ultrastructure of scalariform junctions in the Malpighian tubules of the hemipteran Rhodnius prolixus and the dipteran Aedes taeniorhynchus is described. Both autocellular and intercellular scalariform junctions are illustrated. This is the first report of scalariform junctions in the Malpighian tubules of a dipteran. When combined with previous observations by other authors, the presence of scalariform junctions has now been reported in the Malpighian tubules of insects from five orders, including ametabolous, hemimetabolous, and holometabolous forms. The cell types in which scalariform junctions were found in R. prolixus and A. taeniorhynchus differ in the direction of ion and fluid transport. The cells share the capacity to transport KCl. These same cells also possess morphological features promoting close associations of mitochondria and plasma membranes in the apical region of the cell. The possible role of scalariform junctions is discussed in light of these observations.     

Haematophagy and cleptohaematophagy of clerada apicicornis (Hemiptera: lygaeidae), a potential biological control agent of rhodnius prolixus (Hemiptera: reduviidae)

Because of its ability to prey on Triatominae in rural houses, Clerada apicicornis has been suggested as a potential biological control agent of Rhodnius prolixus. It has also been suggested as a potential vector of mammalian trypanosomes such as Trypanosoma cruzi, because of its ability to take blood directly from mammals. To help resolve these conflicting ideas, we assessed the haematophagic behaviour of C. apicicornis by carrying out feeding trials on laboratory animals. Cleptohaematophagic behaviour was also assessed by allowing C. apicicornis to feed on R. prolixus previously engorged with avian blood. The low proportion of blood meals taken directly from laboratory animals indicates a facultative haematophagy in this species, whereas a greater proportion of nymphs and adults were able to obtain vertebrate blood by predation on engorged R. prolixus. The results suggest that C. apicicornis is unlikely to be effective as a biological control agent, but is also unlikely to have a significant role in the transmission of vertebrate pathogens.     

Cytoskeleton elements mediate the inhibition of the (Na++K+)atpase activity by PKC in Rhodnius prolixus malpighian tubules during hyperosmotic shock

In a previous paper, we observed that the specific activity of (Na++K+)ATPase of the isolated Malpighian tubules from Rhodnius prolixus is inhibited by protein kinase C (PKC) during hyperosmotic shock [Arenstein et al., J Membr Biol 146:47-57 [1995]; Caruso-Neves et al., Z Naturforsch 53c:911-917 [1998]). In the present paper, we study the involvement of the cytoskeleton in this process using isolated Malpighian tubules of Rhodnius prolixus. We observed that pre-incubation of the Malpighian tubule cells in hyperosmotic media decreases the specific activity of (Na++K+)ATPase by 90%. This effect was completely reversed when colchicine, which disrupts microtubules, or cytochalasin B, an inhibitor of actin microfilament polymerization, were added to the media in a dose-dependent manner. The maximal reversion was obtained with colchicine 7.0 microM or cytochalasin B 5.0 microM. The simultaneous addition of sphingosine 50 ng/mL, an inhibitor of PKC, to 10 microM colchicine or 5 microM cytochalasin B, in hyperosmotic media, did not change the stimulatory effect of these drugs on the specific activity of (Na++K+)ATPase. On the other hand, the co-incubation of TPA 20 ng/mL, an activator of PKC, to colchicine or cytochalasin B within hyperosmotic media, abolished the stimulatory effect of these drugs on the specific activity of (Na++K+)ATPase to a similar extent as hyperosmotic shock. These results suggest that inhibition of the (Na++K+)ATPase of the isolated Malpighian tubules from Rhodnius prolixus by PKC during hyperosmotic shock is mediated by cytoskeletal elements.     

Serotonin-elicited inhibition of Cl(-) secretion in the rabbit conjunctival epithelium

The effects of serotonin[5-hydroxytryptamine (5-HT)] on the transepithelial electricalproperties of the short-circuited rabbit conjunctiva were examined.With this epithelium, the short-circuit current(I_sc) measures Cl secretion plusan amiloride-resistant Na⁺ absorptive process. Apicaladdition of 5-HT (10 µM) elicited a prompt I_screduction from 14.2 ± 1.2 to 10.9 ± 1.2 µA/cm² and increased transepithelial resistance from0.89 ± 0.05 to 1.03 ± 0.06 k · cm²(means ± SE, n = 21, P < 0.05).Similar changes were obtained with conjunctivae bathed withoutNa⁺ in the apical bath, as well as with conjunctivaepreexposed to bumetanide with the Cl-dependentI_sc sustained by the parallel activities ofbasolateral Na⁺/H⁺ andCl/HCO exchangers. In contrast, the5-HT-evoked effects were attenuated by the absence of Cl(I_sc = 0.5 ± 0.2, n = 5), suggesting that reduced Clconductance(s) is an effect of 5-HT exposure. In amphotericin B-treatedconjunctiva and in the presence of a transepithelial K⁺gradient, 5-HT addition reduced K⁺ diffusion across thepreparation by 13% and increased transepithelial resistance by 4%(n = 6, P < 0.05), indicating that aninhibition in K⁺ conductance(s) was also detectable.Significant electrical responses also occurred under physiologicalconditions when 5-HT was introduced to epithelia pretreated withadrenergic agonists or protein kinase C, phospholipase C,phosphodiesterase, or adenylyl cyclase inhibitors or after perturbationof Ca²⁺ homeostasis. Briefly, the conjunctiva harbors theonly known Cl-secreting epithelium in which 5-HT evokesCl transport inhibition; receptor subtype and signaltransduction mechanism were not determined.

     

Direct block of the cystic fibrosis transmembrane conductance regulator Cl(-) channel by niflumic acid

Niflumic acid is widely used to inhibit Ca(2+) -activated Cl(-) channels. However, the chemical structure of niflumic acid resembles that of diphenylamine-2-carboxylate, a drug that inhibits the cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. To investigate how niflumic acid inhibits CFTR Cl(-) channel, we studied recombinant wild-type human CFTR in excised inside-out membrane patches. When added to the intracellular solution, niflumic acid caused a concentration- and voltage-dependent decrease of CFTR Cl(-) current with half-maximal inhibitory concentration (K(i)) of 253 microM and Hill co-efficient of approximately 1, at -50 mV. Niflumic acid inhibition of single CFTR Cl(-) channels was characterized by a very fast, flickery block that decreased dramatically current amplitude without altering open-probability. Consistent with these data, spectral analysis of CFTR Cl(-) currents suggested that channel block by niflumic acid was described by the closed <--> open <--> blocked kinetic scheme with blocker on rate (k(on)) = 13.9 x 10(6) M(-1)s(-1), off rate (k(off))=3348 s(-1) and dissociation constant (K(d)) = 241 microM, at -50 mV. Based on these data, we tested the effects of niflumic acid on transepithelial Cl(-) secretion and cyst growth using type I MDCK epithelial cells. Niflumic acid (200 microM) inhibited cAMP-stimulated, bumetanide-sensitive short-circuit current by 55%. Moreover, the drug potently retarded cyst growth. We conclude that niflumic acid is an open-channel blocker of CFTR that inhibits Cl(-) permeation by plugging the channel pore. It or related agents might be of value in the development of new therapies for autosomal dominant polycystic kidney disease.     

Preliminary SAR studies on non-apamin-displacing 4-(aminomethylaryl)pyrrazolopyrimidine K(Ca) channel blockers

An exploratory SAR study on a series of potent, non-apamin-displacing 4-(aminomethylaryl)pyrazolopyrimidine K(Ca) channel blockers is described and their selectivity against K(Ca) channel subtypes is reported. The most potent analog, 5-chloro-N-(thiophen-2-ylmethyl)pyrazolo[1,5-a]pyrimidin-7-amine (24) displayed sub-micromolar activity in both a thallium flux and whole-cell electrophysiology assay and did not displace apamin in a competitive binding study.     

Sites of protein kinase A activation of the human ClC-2 Cl(-) channel

Human ClC-2 Cl(-) (hClC-2) channels are activated by protein kinase A (PKA) and low extracellular pH(o). Both of these effects are prevented by the PKA inhibitor, myristoylated PKI. The aims of the present study were to identify the PKA phosphorylation site(s) important for PKA activation of hClC-2 at neutral and low pH(o) and to examine the relationship between PKA and low pH(o) activation. Recombinant hClC-2 with point mutations of consensus phosphorylation sites was prepared and stably expressed in HEK-293 cells. The responses to forskolin plus isobutylmethylxanthine at neutral and acidic pH(o) were studied by whole cell patch clamp in the presence and absence of phosphatase inhibitors. The double phosphorylation site (RRAT655(A) plus RGET691(A)) mutant hClC-2 lost PKA activation and low pH(o) activation. Either RRAT or RGET was sufficient for PKA activation of hClC-2 at pH(o) 7.4, as long as phosphatase inhibitors (cyclosporin A or endothal) were present. At pH(o) 6 only RGET was needed for PKA activation of hClC-2. Low pH(o) activation of hClC-2 Cl(-) channel activity was PKA-dependent, retained in RGET(A) mutant hClC-2, but lost in RRAT(A) mutant hClC-2. RRAT655(D) mutant hClC-2 was constitutively active and was further activated by PKA at pH(o) 7.4 and 6.0, consistent with the above findings. These results show that activation of hClC-2 is differentially regulated by PKA at two sites, RRAT655 and RGET691. Either RRAT655 or RGET691 was sufficient for activation at pH(o) 7.4. RGET, but not RRAT, was sufficient for activation at pH(o) 6.0. However, in the RGET691(D) mutant, there was PKA activation at pH(o) 6.0.     

Cl(-)/HCO(3)(-) exchange is acetazolamide sensitive and activated by a muscarinic receptor-induced [Ca(2+)](i) increase in salivary acinar cells

Large volumes of saliva are generated by transepithelial Cl(-) movement during parasympathetic muscarinic receptor stimulation. To gain further insight into a major Cl(-) uptake mechanism involved in this process, we have characterized the anion exchanger (AE) activity in mouse serous parotid and mucous sublingual salivary gland acinar cells. The AE activity in acinar cells was Na(+) independent, electroneutral, and sensitive to the anion exchange inhibitor DIDS, properties consistent with the AE members of the SLC4A gene family. Localization studies using a specific antibody to the ubiquitously expressed AE2 isoform labeled acini in both parotid and sublingual glands. Western blot analysis detected an approximately 170-kDa protein that was more highly expressed in the plasma membranes of sublingual than in parotid glands. Correspondingly, the DIDS-sensitive Cl(-)/HCO(3)(-) exchanger activity was significantly greater in sublingual acinar cells. The carbonic anhydrase antagonist acetazolamide markedly inhibited, whereas muscarinic receptor stimulation enhanced, the Cl(-)/HCO(3)(-) exchanger activity in acinar cells from both glands. Intracellular Ca(2+) chelation prevented muscarinic receptor-induced upregulation of the AE, whereas raising the intracellular Ca(2+) concentration with the Ca(2+)-ATPase inhibitor thapsigargin mimicked the effects of muscarinic receptor stimulation. In summary, carbonic anhydrase activity was essential for regulating Cl(-)/HCO(3)(-) exchange in salivary gland acinar cells. Moreover, muscarinic receptor stimulation enhanced AE activity through a Ca(2+)-dependent mechanism. Such forms of regulation may play important roles in modulating fluid and electrolyte secretion by salivary gland acinar cells.     

Dynamic control of cystic fibrosis transmembrane conductance regulator Cl(-)/HCO3(-) selectivity by external Cl(-)

HCO(3)(-) secretion is a vital activity in cystic fibrosis transmembrane conductance regulator (CFTR)-expressing epithelia. However, the role of CFTR in this activity is not well understood. Simultaneous measurements of membrane potential and pH(i) and/or current in CFTRexpressing Xenopus oocytes revealed dynamic control of CFTR Cl(-)/HCO(3)(-) permeability ratio, which is regulated by external Cl(-) (Cl(-)(o)). Thus, reducing external Cl(-) from 110 to 0-10 mm resulted in the expected increase in membrane potential, but with no corresponding OH(-) or HCO(3)(-) influx. Approximately 3-4 min after reducing Cl(o)(-) to 0 mm, an abrupt switch in membrane potential occurs that coincided with an increased rates of OH(-) and HCO(3)(-) influx. The switch in membrane permeability to OH(-)/HCO(3)(-) can also be recorded as a leftward shift in the reversal potential. Furthermore, an increased rate of OH(-) influx in response to elevating pH(o) to 9.0 was observed only after the switch in membrane potential. The time to switch increased to 11 min at Cl(o)(-) of 5 mm. Conversely, re-addition of external Cl(-) after the switch in membrane potential did not stop HCO(3)(-) influx, which continued for about 3.9 min after Cl(-) addition. Importantly, addition of external Cl(-) to cells incubated in Cl(-)-free medium never resulted in HCO(3)(-) efflux. Voltage and current clamp experiments showed that the delayed HCO(3)(-) transport is electrogenic. These results indicate that CFTR exists in two conformations, a Cl(-) only and a Cl(-) and OH(-)/HCO(3)(-) permeable state. The switch between the states is controlled by external Cl(-). Accordingly, a different tryptic pattern of CFTR was found upon digestion in Cl(-)-containing and Cl(-)-free media. The physiological significance of these finding is discussed in the context of HCO(3)(-) secretion by tissues such as the pancreas and salivary glands.     

Bimodal control of a Ca(2+)-activated Cl(-) channel by different Ca(2+) signals

Ca(2+)-activated Cl(-) channels play important roles in a variety of physiological processes, including epithelial secretion, maintenance of smooth muscle tone, and repolarization of the cardiac action potential. It remains unclear, however, exactly how these channels are controlled by Ca(2+) and voltage. Excised inside-out patches containing many Ca(2+)-activated Cl(-) channels from Xenopus oocytes were used to study channel regulation. The currents were mediated by a single type of Cl(-) channel that exhibited an anionic selectivity of I(-) > Br(-) > Cl(-) (3.6:1.9:1.0), irrespective of the direction of the current flow or [Ca(2+)]. However, depending on the amplitude of the Ca(2+) signal, this channel exhibited qualitatively different behaviors. At [Ca(2+)] < 1 microM, the currents activated slowly upon depolarization and deactivated upon hyperpolarization and the steady state current-voltage relationship was strongly outwardly rectifying. At higher [Ca(2+)], the currents did not rectify and were time independent. This difference in behavior at different [Ca(2+)] was explained by an apparent voltage-dependent Ca(2+) sensitivity of the channel. At +120 mV, the EC(50) for channel activation by Ca(2+) was approximately fourfold less than at -120 mV (0.9 vs. 4 microM). Thus, at [Ca(2+)] < 1 microM, inward current was smaller than outward current and the currents were time dependent as a consequence of voltage-dependent changes in Ca(2+) binding. The voltage-dependent Ca(2+) sensitivity was explained by a kinetic gating scheme in which channel activation was Ca(2+) dependent and channel closing was voltage sensitive. This scheme was supported by the observation that deactivation time constants of currents produced by rapid Ca(2+) concentration jumps were voltage sensitive, but that the activation time constants were Ca(2+) sensitive. The deactivation time constants increased linearly with the log of membrane potential. The qualitatively different behaviors of this channel in response to different Ca(2+) concentrations adds a new dimension to Ca(2+) signaling: the same channel can mediate either excitatory or inhibitory responses, depending on the amplitude of the cellular Ca(2+) signal.     

Evidence of a functional CFTR Cl(-) channel in adult alveolar epithelial cells

The cystic fibrosis transmembrane conductance regulator (CFTR) is expressed in the fetal lung, but during lung development it gradually disappears in cells of future alveolar spaces. Recent studies have implicated the CFTR in fluid transport by the adult alveolar epithelium, but its presence has not been demonstrated directly. This study re-evaluated CFTR expression and activity in the adult pulmonary epithelium by using freshly isolated rat alveolar type II (ATII) cells. CFTR mRNA was detected by semiquantitative polymerase chain reaction on the day of cell isolation but was rapidly reduced by 60% after 24 h of cell culture. This was paralleled by a similar decrease of surfactant protein A expression and alkaline phosphatase staining, markers of the ATII cell phenotype. CFTR expression increased significantly on day 4 in cells grown on filters at the air-liquid interface compared with cells submerged or grown on plastic. Significantly higher CFTR expression was detected in distal lung tissue compared with the trachea. The CFTR was also found at the protein level in Western blot experiments employing lysates of freshly isolated alveolar cells. Whole cell patch-clamp experiments revealed cAMP-stimulated, 5-nitro-2-(3-phenylpropylamino)-benzoate-sensitive Cl(-) conductance with a linear current-voltage relationship. In cell-attached membrane patches with 100 microM amiloride in pipette solution, forskolin stimulated channels of approximately 4 pS conductance. Our results indicate that 50-250 of functional CFTR Cl(-) channels occur in adult alveolar cells and could contribute to alveolar liquid homeostasis.     

Possible roles of ENaC and Cl(-) channel in wound closure in Xenopus laevis embryos

Our previous report showed that rapid wound closure in Xenopus laevis embryos was associated with a decrease in the extracellular concentration of either Na(+) or Cl(-) ions. In this study, we examined the wound closure in Xenopus embryos when epithelial Na(+) channel (ENaC), Na(+)/K(+) ATPase (Na(+) pump) or CICs (members of Cl(-) channel) were blocked by each specific inhibitor. Blockage of ENaC and CIC restricted the rate of wound closure during the first 30 min PW and during the subsequent period, respectively. In contrast, inhibition of Na(+) pump had no effect on the rate of wound closure. Furthermore, simultaneous administration of both ENaC and CIC inhibitors resulted in the cumulative reduction of wound closure. Thus, it is plausible that these ion channels play active roles in wound closure in Xenopus embryos. NPPB is known to inhibit both CIC-2 and CIC-3. Immunohistochemical experiments showed that CIC-3, but not CIC-2, was expressed in Xenopus embryos, suggesting that the reduced wound closure by NPPB was due to blockage of CIC-3. A local enhancement of CIC-3 expression at the leading edge of the wounded epidermis was found to be specific to closing wounds that were kept in 10% NAM. An in vitro wounding assay also showed a pattern of CIC-3 expression at the margin of the scratch wound comparable to the results in vivo. These findings suggest that intracellular translocation of CIC-3 is involved in wound closure. We propose that the ion channels, including CIC-3, play a crucial role in wound closure in Xenopus embryos.     

CaMKII inhibition hyperpolarizes membrane and blocks nitrergic IJP by closing a Cl(-) conductance in intestinal smooth muscle

The ionic basis of nitrergic "slow'" inhibitory junction potential (sIJP) is not fully understood. The purpose of the present study was to determine the nature and the role of calmodulin-dependent protein kinase II (CaMKII)-dependent ion conductance in nitrergic neurotransmission at the intestinal smooth muscle neuromuscular junction. Studies were performed in guinea pig ileum. The modified Tomita bath technique was used to induce passive hyperpolarizing electrotonic potentials (ETP) and membrane potential change due to sIJP or drug treatment in the same cell. Changes in membrane potential and ETP were recorded in the same smooth muscle cell, using sharp microelectrode. Nitrergic IJP was elicited by electrical field stimulation in nonadrenergic, noncholinergic conditions and chemical block of purinergic IJP. Modification of ETP during hyperpolarization reflected active conductance change in the smooth muscle. Nitrergic IJP was associated with decreased membrane conductance. The CAMKII inhibitor KN93 but not KN92, the Cl(-) channel blocker niflumic acid (NFA), and the K(ATP)-channel opener cromakalim hyperpolarized the membrane. However, KN93 and NFA were associated with decreased and cromakalim was associated with increased membrane conductance. After maximal NFA-induced hyperpolarization, hyperpolarization associated with KN93 or sIJP was not seen, suggesting a saturation block of the Cl(-) channel signaling. These studies suggest that inhibition of CaMKII-dependent Cl(-) conductance mediates nitrergic sIJP by causing maximal closure of the Cl(-) conductance.     

Mechanism of glucocorticoid-mediated reversal of inhibition of Cl(-)/HCO(-)(3) exchange during chronic ileitis

In the normal ileum, coupled NaCl absorption occurs via the dual operation of Na(+)/H(+) and Cl(-)/HCO(-)(3) exchange on the brush-border membrane (BBM) of villus cells. In a rabbit model of chronic small intestinal inflammation we determined the cellular mechanism of inhibition of NaCl absorption and the effect of steroids on this inhibition. Cl(-)/HCO(-)(3) but not Na(+)/H(+) exchange was reduced in the BBM of villus cells during chronic ileitis. Cl(-)/HCO(-)(3) exchange was inhibited secondary to a decrease in the affinity for Cl(-) rather than an alteration in the maximal rate of uptake of Cl(-) (V(max)). Methylprednisolone (MP) stimulated Cl(-)/HCO(-)(3) exchange in the normal ileum by increasing the V(max) of Cl(-) uptake rather than altering affinity for Cl(-). MP reversed the inhibition of Cl(-)/HCO(-)(3) exchange in rabbits with chronic ileitis. However, MP alleviated the Cl(-)/HCO(-)(3) exchange inhibition by restoring the affinity for Cl(-) rather than altering the V(max) of Cl(-) uptake. These data suggest that glucocorticoids mediate the alleviation of Cl(-)/HCO(-)(3) exchange inhibition in chronically inflamed ileum by reversing the same mechanism that was responsible for inhibition of this transporter rather than exerting a direct effect on the transporter itself, as was the case in normal ileum.